More than two dozens of human hereditary disorders are caused by uncontrollable expansions of simple DNA repeats within various genes. This proposal is to study replication of expandable DMA repeats in vivo and the role of DNA replication in their expansions. During the previously funded period, we have developed a novel strategy for cloning of long non-interrupted repeats in yeast and studied their replication, using electrophoretic analysis of replication intermediates. We have discovered that progression of the replication fork through expandable trinucleotide repeats is compromised. We have further observed that mutations in the replication apparatus, that affect the lagging strand DNA synthesis, can increase the rate of repeat expansions. We have also found that repeats' replication and their propensity to expand can depend on their orientation relative to replication origins. Our working hypothesis is that replication stalling at a repetitive run is the primary event, which could result in the repeat's instability during the replication fork attempts to escape from the stall site. In this proposal, we will assess this hypothesis in both yeast and mammalian systems. We will continue studying replication of expandable repeats, including those, differing from triplet repeats, to affirm that replication stalling is the universal phenomenon for this class of DNA sequences. We will further develop an experimental system, which would allow us to correlate replication stalling with large-scale expansions for various DNA repeats in yeast chromosomal environment. We will exploit this system to unravel the role of cis-acting factors, such as replication origins, in repeat expansions. We will finally extend these studies into mammalian system. We will analyze replication of episomes, which carry expandable repeats, in cultured mammalian cells by various approaches. We will further investigate the length polymorphism and fragility of expandable repeats, replicating in mammalian episomes. Our long-term goal is to understand the mechanisms of repeat expansions in humans.